Nanopores offer a unique capability of sensing and manipulating single molecules in a label-free manner. In a typical nanopore measurement, an insulating membrane separates two chambers containing an electrolyte solution, and analyte molecules in the solution are electrophoretically driven across the barrier via a nanometer-scale aperture contained in the membrane. A characteristic transient drop in the ionic conductance of the pore is observed for each passing molecule, which is used to determine its identity. Over the past decade, nanopore-based techniques have been suggested for a wide range of biophysical and biomedical applications, including DNA sequencing,1,2 RNA sequencing,3 protein sequencing,4-6 drug discovery;7 single-molecule biophysics;8, 9 and proteomics.10-13 
Due to the stochastic nature of single-molecule detection using nanopores, many discrete molecular observations are required in order to obtain statistically significant data for a sample. Multiplexed detection from an array of sensors can considerably speed up measurements, thereby reducing the molecular/biological sample requirement. Furthermore, the ability to introduce sensors tailored for different molecules on a single device can afford complex mixture analysis at unprecedentedly small volumes. However, a critical requirement for this is that each pore in the sensor array is monitored independently, which in the case of electrical detection requires advanced microfluidics and integrated circuitry. Indeed, various schemes have been proposed and demonstrated for multiplexed detection, which include optical approaches,14-19 field effect/tunneling based detection,20-26 and fluid wells connected to electrode arrays.27-29 Nonoptical approaches to reading multiple pores, namely, tunneling-based or fluid wells, are both limited by the need for a network of parallel electrodes and/or fluid conduits that lead to macroscale contacts. On-board amplifiers can alleviate the space requirements of integration, although a recent review estimated that a cost-effective integration would be limited to 1000 amplifiers in a 600 mm2 chip area.30 For comparison, in the Ion Torrent device, a similar sized chip can accommodate a million measurement chambers, three orders-of-magnitude higher than on-board amplified nanopore circuits. Moreover, such nanopore array systems are comprised of two array chips that require alignment, one for circuitry and another for fluidics. Therefore, despite recent demonstrations of devices with arrays of 16 R-hemolysin nanopores,27 16 glass nanopore channels,28 and an 8-channel R-hemolysin platform,29 scaling up of the nanosensor and its readout is more space-consuming than the sensor itself.
In contrast, optical methods for multiplexed detection have made it possible to simultaneously observe optical signals in nanopores using labeled molecules.14-19 However, the need for labeling the sample is restrictive, and detection is plagued with false negatives due to sample bleaching and imperfect labeling. Recently, a method was developed for monitoring ion flow through individual protein channels.32-34 In this method, Ca2+-sensitive fluorescent dyes are used to monitor changes in Ca2+ concentration in the immediate vicinity of membrane channels. Theoretical studies on ion channels have suggested that Ca2+-based approaches can yield signal-to-noise ratios>10:1 at a millisecond time resolution.35 Parallel optical readout of multipore ionic currents at these time resolutions is attractive for emerging nanopore applications, particularly for enzyme-driven DNA sequencing applications.36-39 For these reasons, this approach has been used for localizing and imaging ionic current through multiple ion channel proteins simultaneously.40 Although this appears to be a viable strategy for parallelization of nanopore measurements, the only attempt to utilize the fluorescent sensing of ionic current through nanopores was made by Heron and co-workers.41 However, this study provided only limited insight into the feasibility of the optical detection of ionic current in nanopore experiments, as no biomolecular translocation data were reported, optical imaging was performed at only 100 fps, and the high Ca2+ concentrations used were incompatible with most enzymatic applications. Finally, the approach was limited to lipid-embedded protein channels in a total-internal reflection fluorescence (TIRF) mode, which sets restrictions on the pore size range and the geometry of the setup.
Accordingly, there is a need for systems and methods that allow detection of ion flux through nanopores as a means of analyzing biopolymers. In particular, it would be advantageous for such systems and methods to avoid a requirement for electrically monitoring ionic currents at each pore of the device.